This invention relates generally to tumor-associated antigens, specifically to an antigen found in the urine of cancer patients which can be used for immunodiagnosis, immunoprognosis, and therapy of human cancer.
It is well documented in animal models that cells were changed biochemically and morphologically after neoplastic transformation in vivo. Such transformed neoplastic cells in appropriate doses are capable of inducing protective immunity against tumor development in syngeneic animals when subsequently inoculated with adequate number of viable neoplastic (cancer) cells. The protective immunity was determined to be due to emergence of certain new components that were called tumor-specific transplantation antigens. Expression of similar components, so called tumor-associated antigens, by human malignant tumor cells has been identified by serologic analysis using autologous and allogeneic human sera as the source of antibody. Use of sera from animals immunized with human cancer cells and murine monoclonal antibodies developed against human tumors have added to the definition of additional tumor-associated antigens. However, xeno-polyclonal or murine monoclonal antibody defined antigens on human tumor cells are not necessarily immunogenic in humans. While the physico-chemical properties of almost all of the antigens defined by xeno-polyclonal and murine monoclonal antibodies have been elucidated in great detail, such information is available for only a very few tumor-associated antigens defined by autologous and allogeneic antibodies. The reasons for such paucity have been difficulties in solubilizing adequate amount of the antigens for subsequent purification to homogeneity and the polyclonal nature of autologous and allogeneic antibodies. Without the availability of well characterized tumor-associated antigens that are immunogenic in the host, the importance of these antigens is not fully realized in terms of their application for immunodiagnosis, immunoprognosis and treatment of human cancer.
The tumor-associated antigens in human neoplasms that have been defined by autologous and allogeneic antibodies vary in their distributions. Some are expressed only by individual tumor cell lines or tumors; some are shared by histologically dissimilar tumors including organs where the tumor arises and fetal tissues. The antigens that are expressed only by individual tumors are of limited importance for immunodiagnosis and treatment because tumor cell lines generally do not establish from every single tumor and cannot be applied to another patient. On the contrary, tumor antigens that are shared by different tumors of the same histologic type or by histologically dissimilar tumors can be applied for immunodiagnosis, immunoprognosis and treatment of different patients with different types of cancers.
There are well documented instances to suggest that immunity against growing neoplasm in humans can be enhanced by active immunization with antigen bearing tumor cells. The purpose of such active specific immunotherapy is directed at attempts to enhance the level of anti-tumor immunity beyond that which is naturally induced by the growing neoplasm. It is believed that a growing neoplasm does not induce a maximum immune response in the host to the tumor associated antigens it contains. Most immunotherapy attempts have involved vaccines prepared from whole tumor cells, because progress has been slow in the isolation and purification of human tumor associated antigens. The possibility that living autologous tumor cells could result in tumor growth at the inoculation site has inhibited the use of such vaccines in man. However, tumor cells that express high levels of shared common tumor-associated antigens can be used to immunize different patients (Morton, D. L. et al, In Terry, W. D., Rosenberg, S. A. (eds): Immunotherapy of Human Cancer. New York, Elsevier North Holland, pp 245-249 (1982); Livingston P.O., et al., Int. J. Cancer 31:567 (1983)). The advantage of using such an allogeneic vaccine is two-fold: (1) an immune response induced against the foreign HLA transplantation antigens on the allogeneic vaccinated tumor cells would cause their rejection; (2) this immunization should induce a strong immune response against the shared common cross-reacting tumor-associated antigens to which the human leukocyte antigens (HLA) might serve as a helper function.
Most attempts at immunotherapy in man have been with vaccines composed of inactivated tumor cells, crude extracts or preparations from isolated membranes. Although such preparations may be effective in eliminating progressive tumor growth, there is always the great danger of inactivating the tumor-associated antigens during preparation of the vaccine, unless immunologic reagents and sensitive techniques are available for systemically following the isolation-purification procedure.
Cancer patients who are most likely to respond to an active specific immunotherapy are those who are early in their disease and have minimal residual tumor burden following treatment with other therapeutic modalities (Morton D. L., Seminars in Oncology 13:180 (1986)).
Using lyophilized and reconstituted urine samples of cancer patients and autologous serum as the source of antibody in a complement fixation assay, immunologic reactivity has been observed. The reactivity was abolished by absorption of the sera with tumor cells and not by human normal cells. These observations indicated that immunologically similar antigens were present in the urine samples and tumor cells. Furthermore, the observed reactivity in urine samples of cancer patients who were studied sequentially disappeared after surgical ablation of tumor but reappeared before tumor recurrence, (Gupta, R. K. et al., J. Surg. Oncol., 11:65 (1979)). Because many of the test samples were highly anti-complementary, perhaps due to artifacts arising during lyophilization process, a different method was developed for preparing urine samples for testing. In this study, 24 hour urine samples were obtained from larger numbers of cancer patients and normal controls. The urine samples were concentrated 100-fold by centrifugation and ultrafiltration, and tested by complement fixation using autologous serum as the source of antibody. Ninety-two percent (55/60) of cancer patients were positive for the antigens in their urine as opposed to only 7% (2/27) normal controls. Antibody activity of the sera reacting to the urine from cancer patients was removed by absorption with biopsied tumor specimens but not with normal skin or muscle suggesting that the antigens detected in urine of cancer patients were tumor associated (Rote, N. S. et al, J. Surg. Res. 29:18 (1980).
In a subsequent investigation, an allogeneic serum that had high titer to antigens in urine was used as the source of antibody. Use of this antibody source in complement fixation revealed that urine samples of 94.7% of cancer patients and 35.1% of normal controls were positive. Again, absorption of the allogeneic serum with tumor cells (autologous to the urine source) removed the antibody activity. However, human normal lymphocytes, skin and muscle cells were ineffective as absorbents. Furthermore, excretion of antigens into urine appeared to depend on the presence of tumor in the patients, because removal of tumor by curative surgery resulted in cessation of the putative antigens excretion. The urine remained negative as long as the patient was free of tumor (Rote, N. S. et al., Int. J. Cancer 26:203 (1980)). However, presence of the antigens in 35% of normal urine indicated a cross-reacting antigenic system which prevented this test from practical use.
Gel filtration chromatography of the concentrated urine revealed that the antigenic activity was present in the first peak of the elution profile. However, when this procedure was performed in the presence of 6M urea, the antigenic activity was found in three different peaks representing various molecular sizes, the majority of the activity being in the first peak. However, because of the polyclonal nature of the allogeneic serum that was used as the source of antibody, it was impossible to determine if the antigenic activity in different peaks represented disassociation product of a large antigenic complex bearing the same epitope or represented different epitopes. Similar results were observed when lyophilized and reconstituted urine was used (Rote, N. S. et al, supra). Thus the antigens in urine of cancer patients recognized by autologous and allogeneic antibody was actually a macromolecular complex and because of polyclonal nature of the antibody, the nature of the specific epitope could not be determined. However, the majority of the evidence suggested that the excretion of antigenic macromolecular complex into urine of cancer patients was dependent on the presence of tumor in the cancer host. Serial measurements of tumor-associated antigens in the urine of cancer patients who received preoperative chemo- and radiation therapy were made by complement fixation. The level of excretion of the antigens into urine as a result of therapy were compared to pretreatment samples and changes were correlated with clinicopathological evidence of in situ tumor cell destruction. Of the 53 cancer patient studied in this manner, 44 had clinicopathologic evidence of tumor destruction induced by the preoperative therapy, and all 44 patients had four-fold or greater rise in the level of urinary antigens during the treatment period. The other nine patients had no evidence of tumor destruction and the antigen titers in these patients remained unchanged. These results suggested that excretion of tumor-associated antigens in urine could be used to assess the in vivo effectiveness of tumoricidal therapy of nonaccessible tumors (Huth, J. F. et al., Cancer Treat. Rep. 65:1037 (1981)). Similar results were observed in patients with colon carcinoma receiving hyperthermia and chemotherapy. Again, the incidence of antigenic activity in urine of apparently healthy individuals was high, i.e. 10% (2/20). (Fink, S. J. et al., J. Surg. Oncol. 21:81 (1982)).
Because allogeneic serum was used as the source of antibody in the complement fixation assay, the possibility existed that part of the immunologic reactivity with urine samples could be due to histocompatibility antigens. Therefore, the serum was absorbed with pooled lymphocytes to remove as much anti-HLA antibodies as possible from the serum. This often added anticomplementary activity to the serum. This problem was obviated by using the serum at a dilution beyond the anticomplementary activity level. However, this resulted in reduced sensitivity of the assay. Furthermore, some of the test (urine) samples by themselves exhibited the anticomplementary activity rendering them unsuitable for detection of urinary antigens by complement fixation. To circumvent these problems, a competitive inhibition enzyme immunoassay was developed. In this assay, reactivity between known amounts of autologous antibody and tumor-associated urinary antigens was competitively inhibited by allogeneic urine (test) samples only if the test samples contained immunologically similar antigens. The results of the assay correlated very well with the results of complement fixation without having to deal with the problem of anticomplementary activity and reactivity due to HLA present in the test urine samples. However, the test lacked specificity because of reactivity with urine of normal individuals. (Huth, J. F. et al., Cancer 47:2856 (1981)).
Analysis of urinary tumor-associated antigens by gel-filtration chromatography consistently revealed that the antigenic complex recognized by autologous and allogeneic antibodies had a molecular mass of greater than 300 kD. This antigenic mass was clearly too large to pass through the glomerular basement membrane of kidney by simple diffusion. There are several reports in literature concerning the development of nephrotic syndrome in cancer patients. Renal biopsies of these patients often demonstrated the deposition of immune complexes within the glomerular basement membrane (Laughridge, L. W. and Lewis, M. G., Lancet 1:256 (1971); Couser, W. G. et al., Am. J. Med. 57:962 (1974)). Thus, it was logical to assume that antigens shed by tumor cells in vivo into circulation would react with specific antibodies to form circulating immune complexes. These immune complexes might deposit in the glomerular basement membrane and cause membrane damage that would allow the passage of high molecular weight antigenic complex into the urine. A relationship between antigen nonspecific immune complexes in circulation of cancer patients and excretion of urinary antigens was observed. Of 36 cancer patients who were positive for urinary antigens, 28 (78%) were also positive for circulating immune complexes at the time of urine collection. Of 24 patients that were negative for circulating immune complexes, 22 (92%) were also negative for urinary antigens. In a cancer patient whose serum and urine samples were studied sequentially during this course of thermochemotherapy, fluctuations in the levels of circulating immune complexes and excretion of urinary antigens were parallel. These results suggested that excretion of urinary tumor-associated antigens into urine of cancer patients was not an isolated phenomenon; rather, immune complex deposition in kidneys appeared to cause glomerular damage which allowed passage of the antigens into the urine, Huth, J. F. et al., Cancer 49:1150, (1982).
In an attempt to determine the applicability of the urinary tumor-associated antigens for prognostication of cancer patients, the antigenic complex was partially purified and used as target antigen in the competitive inhibition enzyme immunoassay. One hundred-fold concentrated urine samples from normal controls and melanoma patients were used to establish the base line, distribution of the antigens, and early detection of subclinical recurrence. The results were expressed as antigen units (ng antigenic protein/mg creatinine/24 hours) for comparison among individuals. The antigen levels in urine of melanoma patients (median=56.5 units, n=56) were significantly higher (p&lt;0.05) than those of normal controls (median=1.9 units, n=56). The 90th percentile for the normal group was 34.3 antigen units. Using this value as the criterion for positivity, 64% (36/56) urine samples of melanoma group were positive for the antigens as opposed to 11% (6/56) of normal controls. Subsequently a retrospective analysis of 58 melanoma patients paired on the basis of disease recurrence and no recurrence after lymphadenectomy revealed a median antigen level of 68 units for the recurrent group and 18.9 for the non-recurrent group. Eighteen of 29 (62%) melanoma patients who had recurrence of their disease and 9 of 29 (31%) patients who remained disease free were urinary antigen positive. These incidences were significantly different (p&lt;0.005) (Gupta, R. K. et al., Diagnostic Immunol. 1:303 (1983)). Though the results of the above investigations confirmed previous observations, the utility of urinary antigen detection assay remained uncertain because of the consistent observation that urine of many (11%) normal individuals had considerable levels of the antigen.
Despite significant progress made in developing assays using tumor markers that are not immunogenic in the cancer host, e.g., CEA, alpha-fetoprotein, prostate specific antigen, etc., there exists a need to diagnose and treat tumors using tumor-associated antigens that are immunogenic in the cancer host. This invention satisfies these needs by providing for the detection of various tumors by detecting U-TAA while avoiding the detection of false positives. In addition, this invention provides antigenic subunits of U-TAA and a vaccine which induces cell mediated specificity for individual determinants on the tumor cell surface, as well as anti-U-TAA antibody production.